System for blood separation by microfluidic acoustic focusing in separation channels with dimensions defined based on properties of standing waves

ABSTRACT

Systems and methods for cleansing blood are disclosed herein. The methods include acoustically separating undesirable particles bound to capture particles from formed elements of whole blood. After introducing the capture particles to whole blood containing undesirable particles, the whole blood and capture particles are flowed through a microfluidic separation channel. At least one bulk acoustic transducer is attached to the microfluidic separation channel. A standing acoustic wave, imparted on the channel and its contents by the bulk acoustic transducer, drives the formed elements and undesirable particles bound to capture particles to specific aggregation axes. After aggregating the particles, the formed elements exit the separation channel through a first outlet and are returned to the patient. The undesirable particles, bound to the capture particles, exit through a second outlet and can be discarded to saved for later study.

RELATED CASES

The present application is a continuation of, and claims priority to,U.S. patent application Ser. No. 14/772,216, filed Sep. 2, 2015, whichis a U.S. National Stage Application of, and claims priority to,International Application No. PCT/US2014/022701, filed on Mar. 10, 2014,which claims priority to U.S. Provisional Patent Application No.61/775,233, filed on Mar. 8, 2013, and titled “System and Method ForBlood Separation by Microfluidic Acoustic Focusing,” which isincorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

Sepsis is a disease with a very significant public health impact thathas stubbornly resisted new therapies. Antibiotics are the only realtherapeutic option, yet sepsis can be caused by over 100 bacteria andmany fungi so a universal antibiotic is not a realistic option; theantibiotics and antifungals used have significant complications and areoften unsuitable for fragile patients. The concept of cleansing theblood has been tried previously without success. Previous bloodcleansing concepts have included laboratory scale methods ofcentrifugation, capillary electrophoresis, liquid chromatography, fieldflow fractionation, and liquid-liquid extraction. These devices havefailed to deliver continuous flow cleansing devices. In additional tooften discarding large portions of the blood, current cleansing devicesmay rely on: diluents, sheath flow, controlled solution conductivity,costly microfabricated on-chip materials, and toxic additives.

SUMMARY OF THE DISCLOSURE

According to one aspect of the disclosure, a blood cleansing deviceincludes a microfluidic separation channel having an upstream portionand a downstream portion wherein the separation channel is shaped suchthat an interior portion of the upstream portion of the separationchannel is substantially aligned with a wall of the downstream portionof the downstream portion of the separation channel. A width of theseparation channel is between about 30% and 45% of a wavelength of astanding wave applied to the separation channel. Additionally, theseparation channel includes a first inlet configured to introduceflowing whole blood into the upstream portion of the separation channel.The whole blood includes plasma, a plurality of formed elements and aplurality of undesirable particles. The separation channel also includesa first outlet positioned downstream from the first inlet and upstreamfrom the downstream portion of the separation channel, a second outletpositioned within the downstream portion of the separation channel alongthe wall of the downstream portion that is aligned with the interiorportion of the upstream portion of the separation channel, a secondinlet positioned within the separation channel between the first outletand the second outlet, and a third outlet positioned in the downstreamportion of the separation channel downstream of the second outlet.

In some implementations, the width of the separation channel is betweenabout 30% and about 35% or between about 35% and 45% of the wavelengthof the standing wave applied to the separation channel. A thickness ofthe wall is between about 25% and about 45% of the wavelength of thestanding wave applied to the separation channel.

In some implementations, the blood cleansing device includes at least asecond separation channel and a third separation channel. The firstoutlet of the second separation channel is configured to carry fluidfrom the second separation channel to the second inlet of the separationchannel, and the second inlet of the third separation channel isconfigured to receive fluid from the first outlet of the separationchannel.

In some implementations, the blood cleansing device includes amicrofluidic injector for introducing, into the separation channel, aplurality of lipid-based capture particles configured to bind to theundesirable particles. In some implementations, the microfluidicinjector includes one of a microfluidic nozzle and a porous membranecoupled to a lipid reservoir. In other implementations, the separationchannel is formed in a substrate comprising one of polystyrene, glass,and polyimide, acrylic, polysulfone, and silicon.

In other implementations, the blood cleansing device includes anacoustic transducer positioned adjacent to the separation channel andconfigured to impose a standing wave transverse to the length of theupstream portion of separation channel. The wave is configured to focusformed elements and a plurality of the undesirable particles bound to aplurality of capture agents towards an interior region of the upstreamportion of the channel. Some implementations include a second acoustictransducer positioned adjacent the downstream portion of the separationchannel and configured to impose a standing wave transverse to thelength of the second portion of the separation channel. The wave fromthe second acoustic transducer is selected such that the formed elementswill be driven away from the wall of downstream portion of theseparation channel at a rate that is faster than a rate at which thewave drives the undesirable particles bound to the capture agents awayfrom the wall.

In some implementations, the blood cleansing device includes a secondoutlet positioned sufficiently downstream within the downstream portionof the separation channel that in operation, a greater percentage of theundesirable particles that are bound to the capture agents flows out ofthe second outlet than out of the third outlet and a greater percentageof the formed elements flows out of the third outlet than out of thesecond outlet.

In some implementations, the upstream portion of the separation channelincludes an aggregation point, and the upstream portion of theseparation channel is configured such that a width of the first portionof the separation channel at the aggregation point is half thewavelength of the acoustic wave acting on the whole blood.

In other implementations, the separation channel includes walls having athickness at a particle aggregation point that is a multiple of onequarter of the wavelength of an acoustic wave acting on the walls of theseparation channel. In some implementations, the height of theseparation channel at a particle aggregation point is less than onequarter of the wavelength of a standing acoustic wave acting on theparticle aggregation point.

According to another aspect of the disclosure, a blood cleansing deviceincludes a microfluidic separation channel having an upstream end anddownstream end. The separation channel includes a first inlet configuredto introduce flowing whole blood into a proximal end of the separationchannel, the whole blood including plasma, a plurality of formedelements and a plurality of undesirable particles. A width of theseparation channel is between about 30% and 45% of a wavelength of astanding wave applied to the separation channel. The separation channelalso includes a first outlet at the downstream end of the separationchannel positioned substantially along the longitudinal axis of theseparation channel, a second outlet at the downstream end positionedadjacent a first wall of the separation channel, and a third outlet atthe downstream end positioned adjacent a second wall of the separationchannel, opposite the first wall. The device also includes an acoustictransducer positioned adjacent to the separation channel for imposingthe standing acoustic wave transverse to a particle migration region ofthe separation channel, and a capture particle injector configured tointroduce a plurality of lipid-based capture particles into the wholeblood before the blood reaches the particle migration region of theseparation channel.

In some implementations, the width of the separation channel is betweenabout 30% and about 35% or between about 35% and 45% of the wavelengthof the standing wave applied to the separation channel. A thickness ofthe wall is between about 25% and about 45% of the wavelength of thestanding wave applied to the separation channel.

In some implementations, the device includes a reservoir in fluidiccommunication with the capture particle injector. In someimplementations, reservoir contains a plurality of the lipid-basedcapture particles. In other implementations, the reservoir contains amixture of materials, which when directed by the capture particleinjector into the whole blood, form the lipid-based capture particles.In these implementations the materials in the mixture include anaffinity molecule, a lipid, and a fluid with a density less than 1g/cm³. In some implementations, the affinity molecule is aglycoconjugate and/or lectin. In some implementations, the lipid-basedcapture particles have significantly different acoustophoretic mobilitythan that of formed elements of blood. In some implementations, thecapture particle injector includes a microfluidic nozzle. In someimplementations, the capture particle injector includes a porousmembrane. In yet other implementations, the second and third outletsmerge at a fourth outlet.

According to one aspect of the disclosure, a method of cleanings bloodincludes flowing whole blood, containing a plurality of undesirableparticles, formed elements, and plasma, through a microfluidicseparation channel. Then injecting capture particles, from a reservoir,into the separation channel such that the capture particles can bind toat least a plurality of the undesirable particles. A width of theseparation channel is between about 30% and 45% of a wavelength of afirst standing wave applied to the separation channel. Next, upstream ofa first outlet of the separation channel, with the first standingacoustic wave, the whole blood and the captures particles are directedaway from the walls of the separation channel. Then the method continueswith directing, downstream of the first outlet and prior to a secondoutlet of the separation channel, the captures particles and the formedelements alongside a wall of the separation channel, and finallydriving, prior to the second outlet, with a second standing acousticwave, formed elements of the flowing whole blood away from the walls ofthe separation channel as the capture particles remain sufficientlyclose to the wall of the separation channel to flow out of the secondoutlet, while the formed elements flow out of a third outlet of theseparation channel.

In some implementations, the width of the separation channel is betweenabout 30% and about 35% or between about 35% and 45% of the wavelengthof the first standing wave applied to the separation channel. Athickness of the wall is between about 25% and about 45% of thewavelength of the first standing wave applied to the separation channel.

In some implementations, the capture particles include affinitymolecules anchored to a lipid bilayer encapsulating a fluid. In someimplementations, the fluid has a density less than about 1 g/cm³.

In some implementations, the affinity molecule, lipid, and fluid aremixed in the reservoir prior to their injection into the separationchannel. In some of these implementations, injecting the captureparticles includes injecting the mixture of the affinity molecule,lipid, and fluid through a nozzle such that the lipid forms a liposomesurrounding the fluid. In some implementations, the capture particleincludes a polystyrene bead. In yet other implementations, the captureparticles have significantly different acoustophoretic mobility thanthat of formed elements of blood. In some implementations, the captureparticles are less about 10 μm and greater than about 2 μm in diameter.

In some implementations, the formed elements of blood include at leastone of red blood cells, white blood cells, and platelets. In someimplementations, the method further includes unbinding the captureparticles from the undesirable particles after flowing through the thirdoutlet and introducing the unbound capture particles into the separationchannel.

In other implementations, the formed elements are driven away from thewalls at a faster rate than the capture particles and undesirableparticles by the standing acoustic wave. In some implementations, themethod further includes extracting whole blood from a patient prior toflowing the whole blood through the separation channel, the extractedwhole blood having a first concentration of undesirable particles, andthen reintroducing whole blood with fewer undesirable particles backinto the patient after flowing whole blood through the plurality ofmicrochannels.

According to another aspect of the disclosure, a method of cleansingblood includes flowing a suspension through a microfluidic separationchannel. A width of the separation channel is between about 30% and 45%of a wavelength of a standing wave applied to the separation channel.The suspension includes a plurality of target particles having apositive acoustic contrast factor and a plurality of undesirableparticles suspended in a fluid. The method also includes introducing,into the suspension, a plurality of positive contrast-factor captureparticles selected to bind to the undesirable particles. The magnitudeof the positive contrast-factor of the capture particles issubstantially different than that of the target molecules. The methodalso includes applying the standing acoustic wave across the separationchannel transverse to the direction of the flow of the suspensionthrough the separation channel such that a pressure node forms along aninterior axis of the separation channel, and collecting thepositive-factor capture particles through an outlet of the separationchannel at a distance along the separation channel that is sufficientlybeyond the point in the channel at which the standing acoustic wave isintroduced that the target particles are separated from thepositive-factor capture particles.

According to another aspect of the disclosure, a method of cleansingblood includes flowing whole blood, including plasma, a plurality offormed elements, and a plurality of undesirable particles, into an inletof a microfluidic separation channel. A width of the separation channelis between about 30% and 45% of a wavelength of the standing waveapplied to the separation channel. The method includes introducing aplurality of lipid-based capture particles into the whole blood suchthat the lipid-based capture particles bind to a plurality of theundesirable particles. The standing acoustic wave is applied transverseto a direction of flow of the whole blood through the separation channelsuch that the formed elements aggregate to about the axial center of theseparation channel and the capture particles aggregate along at leastone wall of the separation channel. The method also includes collectingthe formed elements of the whole blood at a first outlet positioned at adownstream end of the separation channel at about the axial center ofthe separation channel. Then the capture particles are collected throughat least a second outlet positioned at the downstream end of theseparation channel adjacent to the at least one wall along which thecapture particles are aggregated.

In some implementations, the width of the separation channel is betweenabout 30% and about 35% or between about 35% and 45% of the wavelengthof the standing wave applied to the separation channel. A thickness ofthe wall is between about 25% and about 45% of the wavelength of thestanding wave applied to the separation channel.

In some implementations, at least one wall includes at least one sidewall of the separation channel, at least one of the top walls, and/orbottom walls of the separation channel. In some implementations, thecapture particles comprise affinity molecules anchored to a lipidbilayer encapsulating a fluid. In some implementations, the fluid has adensity less than 1 g/cm³. In other implementations, the affinitymolecule, lipid, and fluid are mixed in the reservoir prior to theirinjection into the separation channel. In some implementations,injecting the capture particles includes injecting the mixture of theaffinity molecule, lipid, and fluid through a nozzle such that the lipidforms a liposome surrounding the fluid. In some implementations, thecapture particle includes a polystyrene bead. In some implementations,the capture particles have significantly different acoustophoreticmobility than that of formed elements of blood. In some implementations,the capture particles are less than 5 μm or 2 μm in diameter. In someimplementations, the formed elements of blood includes at least one ofred blood cells, white blood cells, and platelets. In someimplementations, the method further includes, extracting whole bloodfrom a patient prior to flowing the whole blood through the separationchannel, the extracted whole blood having a first concentration ofundesirable particles, and reintroducing whole blood with fewerundesirable particles back into the patient after flowing whole bloodthrough the plurality of microchannels.

According to another aspect of the disclosure, a method of cleansingblood includes flowing a suspension, which includes a plurality oftarget particles and a plurality of undesirable particles suspended in afluid into an inlet of a microfluidic separation channel. A width of theseparation channel is between about 30% and 45% of a wavelength of thestanding wave applied to the separation channel. The method alsoincludes introducing into the suspension a plurality of lipid-basedcapture particles such that the lipid-based capture particles bind to aplurality of the undesirable particles. The standing acoustic wave isapplied transverse to a direction of flow of the suspension through theseparation channel such that the target particles aggregate to about theaxial center of the separation channel and the capture particles alsoaggregate along at least one wall of the separation channel.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the described implementations may be shownexaggerated or enlarged to facilitate an understanding of the describedimplementations. In the drawings, like reference characters generallyrefer to like features, functionally similar and/or structurally similarelements throughout the various drawings. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the teachings. The drawings are not intended to limitthe scope of the present teachings in any way. The system and method maybe better understood from the following illustrative description withreference to the following drawings in which:

FIG. 1 is a block diagram of a system for cleansing blood, according toone illustrative embodiment;

FIG. 2 is a top view of a single-stage separation channel, such as canbe used in the system of FIG. 1 , according to one illustrativeembodiment;

FIG. 3 is a top view of a network of two-stage separation channels, suchas can be used in FIG. 1 , according to one illustrative embodiment;

FIG. 4 is a cross sectional view of a single-stage separation channel,such as the separation channel of FIG. 2 , mounted to a bulk transducer,according to one illustrative embodiment;

FIG. 5A is a cross sectional view a single-stage separation channel, asdepicted in FIG. 2 , containing a plurality of particles lacking anactive acoustic transducer, according to one illustrative embodiment;

FIG. 5B is a cross sectional of a single-stage separation channel, asdepicted in FIG. 2 , containing a plurality of particles adjacent to anactive acoustic transducer, according to one illustrative embodiment;

FIG. 6A is a top view of a separation channel, as depicted in FIG. 2 ,in which fluid is flown through the channel without the application ofthe standing acoustic wave;

FIG. 6B is a top view of a separation channel, as depicted in FIG. 2 ,after the application of a standing acoustic wave, according to oneillustrative embodiment;

FIG. 7 is a cut away of a lipid-based capture particle, according to oneillustrative embodiment;

FIGS. 8A-8E are illustrations of the components and use for a captureparticle, as depicted in FIG. 7 , according to one illustrativeembodiment;

FIG. 9 is a flow chart of a method for cleansing blood with a two-stageseparation channel, as depicted in FIG. 3 , according to oneillustrative embodiment; and

FIG. 10 is a flow chart of a method for cleansing blood with asingle-stage separation channel, as depicted in FIG. 2 , according toone illustrative embodiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The various concepts introduced above and discussed in greater detailbelow may be implemented in any of numerous ways, as the describedconcepts are not limited to any particular manner of implementation.Examples of specific implementations and applications are providedprimarily for illustrative purposes.

The present system and methods described herein generally relates to asystem for cleansing blood. Accordingly, in various implementations, thedisclosure relates to the cleansing of whole blood by acousticallyseparating undesirable particles from the blood via high throughputmicrofluidic arrays. In certain implementations, in part to overcome theprior deficiencies with the poor performance of acoustic separation onsmall particles, prior to acoustic separation of the blood, captureparticles are introduced and mixed with the blood to form complexes withthe undesirable particles, yielding particles large enough to beeffectively and efficiently targeted by acoustic separation.

FIG. 1 illustrates a system 100 for cleansing blood by removing wastematerial such as bacteria, viruses and toxins. In the system 100, bloodis removed from a patient via an intravenous line 102. The blood is thenpumped to a mixing chamber 104 by a first pump 103. In the mixingchamber 104, capture particles are mixed with whole blood. Thecomponents of the capture particles are stored in a reservoir 107. Fromthe reservoir 107, the capture particles are pumped by a second pump 106into the mixing chamber. The capture particles are formed as thecontents of the reservoir 107 are extruded from a micronozzle 105 at theentrance to the mixing chamber 104. From the mixing chamber 104, thewhole blood and capture particles enter a manifold system 107. Themanifold system 107 distributes the whole blood and capture particles toa plurality of separation channels contained within the microfluidicflow chamber 108. The microfluidic flow chamber 108 sits atop at leastone bulk piezoelectric acoustic transducer 109. The acoustic wavesgenerated by the bulk piezoelectric acoustic transducers are used tofunnel the contents of the whole blood and capture particles to specificoutlets of the separation channels. As the whole blood flows through themicrofluidic flow chamber 108, cleansed blood flows to a first outlet110. After exiting the first outlet 100, the cleansed blood returns tothe patient 101, via a second intravenous line 111. The captureparticles and other waste material removed from the blood exit themicrofluidic flow chamber 108 via a second outlet 112. Next, the wastematerial and capture particles enter a waste collection unit 113. In thewaste collection unit 113, the capture particles are separated from thewaste material. Once separated, the waste material is discarded and thecapture particles are returned to the reservoir 107 by tubing 114. Oncereturned to the reservoir 107, the capture particles are reused in thesystem to remove additional waste material from whole blood as itcontinues to flow through the system.

The system 100, as illustrated, includes a pump 103 for moving bloodfrom the patient 101 to the mixing chamber 104. In some implementations,the pump operates continuously, while in other implementations the pumpworks intermittently, and only activates when the level of whole bloodin the mixing chamber 104 or manifold falls below a set threshold. Insome implementations, the flow rate of the pump is configurable, suchthat the rate the blood exits the patient can be configured to be fasteror slower than if no pump was used. In yet other implementations, noexternal pump is required. In this example, the blood is transported tothe mixing chamber 104 by the pressure generated by the patient's ownheart. In some implementations, the patient 101 is connected to a bloodpressure monitor, which in turn controls the pump. Example pumps caninclude, but are not limited, to peristaltic pumps or any other pumpsuitable for flowing blood.

As illustrated in the system 100, capture particles are also pumped intothe mixing chamber. A second pump 106 pumps the ingredients to form thecapture particles from a reservoir 107 to the mixing chamber 104. Insome implementations, the components of the capture particles arecontinuously agitated in the reservoir 107 in order to keep thecomponents well mixed. The components are formed into capture particlesas they enter the mixing chamber 104. The components enter the mixingchamber 104 through a micronozzle 105. In some implementations, themicronozzle 105 injects the capture particles into the mixing chamber104. In other implementations, the micronozzle 105 injects the captureparticles into the manifold system 107, and in yet other implementationsthe micronozzle 105 is positioned such that it injects capture particlesdirectly into the separation channels of the microfluidic chamber 108.In some implementations, the micronozzle 105 is a micro-machined nozzle,configured to allow a specific amount of the capture particle componentsthrough the nozzle at a given time. In some implementations, themicronozzle is an array of micronozzles. In yet other implementations,the micronozzle is a membrane with pores. The pump 106 is configured toflow the contents of the reservoir through the micronozzle 105 at apredetermined rate such that the amphipathic characteristics of themolecules of the components of the captures particles cause the captureparticles to spontaneously form as they exit the micronozzle 105.

In some implementations, a micronozzle is not used to generate thecapture particles. In these implementations, the capture particles arepremade. The capture particles are then stored in the reservoir and thenintroduced into the system by the pump 106 at either the mixing chamber104, manifold system 107, and/or the separation channels of themicrofluidic flow chamber 108.

As illustrated in system 100, the whole blood containing undesirableparticles and the capture particles enter the mixing chamber 104. Insome implantations, the contents of the mixing chamber are continuouslyagitated to improve distribution of the capture particles throughout thewhole blood and undesirable particles such that the capture particlesbind to the undesirable particles. In some implementations,anticoagulants or blood thinners are introduced into the mixing chamber104 to assist the blood as it flows through the system 100. In someimplementations, the mixing chamber 104 contains a heating element forwarming the contents of the mixing chamber 104.

The contents of the mixing chamber 104 then flow into the manifoldsystem 107, as illustrated by system 100. The manifold system 107 flowsthe whole blood, undesirable particles, and capture particles into theinlets of the plurality of separation channels of the microfluidic flowchamber 108.

In the illustrated system 100, the microfluidic flow chamber 108contains a plurality of separation channels. The capture particles andundesirable particles are driven with standing acoustic waves tooutlets. In some implementations, the separation occurs during a singlestage, while in other implementations, the separation occurs over aplurality of stages. In some implementations, the microfluidic flowchamber is disposable.

As show in the illustrations of system 100, the microfluidic flowchamber 108 sits atop a bulk piezoelectric acoustic transducer 109. Insome implementations, the system 100 contains a single bulkpiezoelectric acoustic transducer 109, while in other implementationsthe system 100 contains a plurality of bulk piezoelectric acoustictransducers 109.

In some implementations, the bulk piezoelectric acoustic transducer 109is glued to the microfluidic flow chamber 108. In other implementationsthe microfluidic flow chamber 108 is clamped to the bulk piezoelectricacoustic transducer 109 so the microfluidic flow chamber may easily beremoved from the system. In other implementations the adhesive materialconnecting the bulk piezoelectric acoustic transducer 109 to themicrofluidic flow chamber 108 is removable, for example by heating theadhesive.

The bulk piezoelectric acoustic transducer 109 imposes a standingacoustic wave on the separation channels of the microfluidic flowchamber 108 transverse to the flow of the fluid within the microfluidicflow chamber 108. The standing acoustic waves are used to drive fluidconstituents towards or away from the walls of the separation channelsor other aggregation axes.

More particularly, the dimensions of the separation channels areselected based on the wavelength of the imposed standing wave such apressure node exists at about the center or other interior axis of theseparating channel, while antinodes exists at about the walls of theseparation channel. Particles are driven to different positions withinthe channel based on the sign of their acoustic contrast factor at arate which is proportional to the magnitude of their contrast factor.Particles with a positive contrast factor (e.g. the formed elements ofblood) are driven towards the pressure node within the interior of theseparation channel. In contrast, particles with a negative contrastfactor are driven toward the pressure antinodes. These principles aredepicted and described further in relation to FIGS. 5A and 5B.

Based on these principles, formed elements of blood can be separatedfrom capture particles (and thus the undesirable particles bound to thecapture particles) in two ways. In one way, as described further inrelation to FIGS. 2 and 10 , capture particles are selected to havenegative contrast factors, which is opposite to the positive contrastfactors of the formed elements of blood. Thus, in response to thestanding acoustic wave, the formed elements are driven towards theresulting pressure node while the capture particles are driven towardsthe antinodes.

This technique can be used in a single-stage separation system. As wholeblood, undesirable particles and capture particles mix in the mixingchamber 104 and continue to mix as flowing through the manifold system107, the capture particles bind to the undesirable particles. As thewhole blood, undesirable particles and capture particles enter the areaof the separation channel where the standing acoustic wave is imparted,the standing acoustic wave drives the capture particles and boundundesirable particles to a specific axis (e.g., against the wall of theseparation channel) and the formed elements of the whole blood to asecond axis (e.g., the middle of the separation channel). Thus, thecapture particles and undesirable particles can be collected from theedges of the separation channel and disposed of while the cleaned bloodis collected and returned to the patient.

Alternatively, capture particles can be separated from formed elementsof blood based on a time of flight principle. That is, if the captureparticles are selected to have a contrast factor that is the same signas that of the formed elements of blood, but with a substantiallydifferent magnitude, and assuming the formed elements and captureparticles are substantially aligned prior to the application of astanding wave at a distance away from the positive pressure node inducedby the wave, the formed elements and capture particles will migratetowards the pressure node at different rates. Thus, the formed elementsand capture particles can be collected separately at a point where thehigher contrast factor particles (capture particles or formed elementsdepending on the selected capture particles) have move sufficiently farfrom the initial aggregation axis that they have separated from thecapture particles due to their difference in acoustophoretic mobility.Thus, in some implementations, a two-stage separation process isemployed. In the two-stage process, formed elements of blood and captureparticles are first aggregated along a common first axis of theseparation channel using a first standing acoustic wave. Then after theyhave reached the common aggregation axis, a second standing acousticwave drives the formed elements and capture particles to a secondaggregation axis. However, instead of waiting until the formed elementsand capture particles all reach the second aggregation axis, the channelsplits to direct the particles having the lower acoustophoretic mobilitydown a first outlet. The particles that have a greater acoustophoreticmobility, which would have already migrated towards the secondaggregation axis to a point that they are beyond entrance to the firstoutlet, flow out a second outlet. This separation technique is describedfurther in relation to FIGS. 3 and 10 .

As illustrated in the system 100, the cleansed blood exits themicrofluidic flow chamber 108 at a first outlet 110. From there theblood is returned to the patient 101 via an intravenous supply line 111.In some implementations, the blood in the supply line 111 is reheated tobody temperature before returning to the patient 101. In otherimplementations an infusion pump is used to return the blood to thepatient 101, while in the system 100 the pressure generated in thesystem by pumps 103 and 106 is adequate to force the blood to return tothe patient 101.

As illustrated in the system 100, waste material (e.g. the captureparticle and undesirable particles) exit the microfluidic flow chamber108 and enter a waste collection unit 113. In some implementations, thewaste collection unit 113 contains a capture particle recycler. Thecapture particle recycler unbinds the undesirable particles from thecapture particles. The capture particles are then returned to thereservoir 107 via tubing 114. The undesirable particles are thendisposed of. In some implementations, the undesirable particles aresaved for further testing.

While the system 100 is described above for the in-line cleansing of apatient's blood, in alternative implementations, the system 100 can beused to cleanse stored blood. For example, the system 100 can be used tocleanse collected blood for later infusion to help ensure the safety ofthe blood.

FIG. 2 illustrates an example single-stage separation channel suitablefor use within the microfluidic flow chamber 108 of the blood cleansingsystem 100. The separation channel includes an inlet 202, a flow channel203, a first outlet 204, a first outlet channel 206, a second outletchannel 207, and a second outlet 205. The separation channel ismanufactured in a sheet of material 201.

In FIG. 2 , whole blood, undesirable particles, and capture particlesenter the separation channel at the inlet 202 from the manifold system107. The whole blood, undesirable particles, and capture particles thenflow the length of the flow channel 203. The flow channel is subdividedinto three regions: an upstream region, a downstream region, and amigration region. The migration region lies between the upstream anddownstream regions, and is the region of the flow channel where thestanding acoustic wave is imparted transverse to the flow of particles.As the formed elements of the whole blood, capture particles and theundesirable particles enter the migration region, the standing acousticwave drives the capture particles bound to the undesirable particles tothe side walls of the separation channel, and the formed elements of thewhole blood to the center of the channel. The formed elements of thewhole blood then exit the separation channel through the outlet 204located at about the central axis of the separation channel. The captureparticles and undesirable particles then exit the separation channelthrough the first and second outlet channels 206 and 207 whichterminates in the second outlet 205. In some implementations, the formedelements are driven to the walls of the separation channel and thecapture and undesirable particles remain in the center of the separationchannel.

In some implementations, the separation channel 200 can separateundesirable particles from any fluid. As discussed above and later inrelation to FIGS. 5 and 7 , the separation channel 200 can be used toremove undesirable particles from any fluid, so long as thecharacteristics of the capture particle are appropriately selected. Forexample, selecting an encapsulated fluid such that its density and bulkmodulus gives the capture particle a contrast factor that distinguishesit from the fluid and other particles in the fluid. For example, theseparation channel 200 may be used to remove undesirable particles from,but not limited to, blood plasma, blood serum, water, liquid foodproducts (e.g., milk), and lymph.

In the implementation of FIG. 2 , the outlet 205 is formed from themerging of two outlet channels 206 and 207. In some implementations, thestreams do not rejoin, but lead to separate outlet terminals.

In FIG. 2 , the particles are separated in the same plane as the sheetof material 201 (i.e. particles are aligned to the left, right, orcenter of the channel); however, in other implementations, the particlesare separated out of plane. For example, in some implementations, theparticles are aligned with the top, middle, or bottom of the channel.

In FIG. 2 , the sheet of material 201 can include, but is not limitedto, polystyrene, glass, polyimide, acrylic, polysulfone, and silicon.The channel can be manufactured by a number of manufacturing techniques,including, but not limited to, milling, embossing, and etching.

In some implementations, a higher frequency standing acoustic wave canbe applied to create two pressure nodes within the separation channel200, both spaced apart from the sidewalls of the channel and separatedby an anti-node. In one such implementation, the formed elements in theblood aggregate into two substantially parallel streams near thesidewalls along the pressure nodes, while the capture particles migrateto the center of the channel in line with the anti-node. In suchimplementations, the capture particles exit the separation channelthrough the outlet 204, while the blood exits the separation channelthrough the first and second outlet channels 206 and 207.

FIG. 3 illustrates an example network 300 of multistage separationchannels suitable for use the blood cleansing system 100 depicted inFIG. 1 . The network of separation channels includes a plurality offirst inlets 301. FIG. 3 also includes first and second acoustic bulktransducers 302 and 305, respectively. Additionally, each separationchannel includes an upstream outlet 303 and a second inlet 304. Theupstream outlet 303 of each channel is connected to the second inlet 304of its neighboring channel. The fluid exits the separation channelsthrough a first downstream outlet 307 or second downstream outlet 306.

In each separation channel of the network 300, the flow channel throughwhich most of the fluid in the channel flows shifts after the upstreamoutlet 303. The portion of the separation channel prior to the shift isreferred to as the upstream portion 308 and the portion after the shiftis referred to as the downstream portion 309. As the particles withinthe blood continue to flow down the separation channel, one subset ofparticles is driven into a first downstream outlet 307 and a secondsubset of the particles are driven into a second downstream outlet 306.For example, the cleaned blood exits through the first downstream outlet307 and the capture particles with bound undesirable particles exitsthrough the second downstream outlet 306.

As described above, and illustrated in FIG. 3 , the separation channelsin the network 300 are generally divided into upstream portions 308 anddownstream portions 309. The two portions are distinguished by a shiftin the separation channel. The angle of the shift is referred to as thebranching angle. The branching angle for a particular implementation ischosen to substantially align a wall of the downstream portion 309 withan interior axis of the upstream portion 308. The selected axiscorresponds to the location of a pressure node induced in the channel bythe first acoustic transducer 302. For example, in some implementations,a wall of the downstream portion 309 is configured to align with aninterior axis substantially in the middle of the upstream portion 308.

In the illustration of FIG. 3 , there are a plurality of first inlets301. The number of first inlets, and thus separation channels, can beincreased to n where n is the number of separation channels required tomeet the flow demands of a specific implementation. In someimplementations, the first inlets 301 are configured to accept flowingwhole blood and a plurality of capture particles. The flowing wholeblood includes plasma, a plurality of formed elements and a plurality ofundesirable particles. The formed elements of blood can includeleukocytes (white blood cells), erythrocytes (red blood cells), andthrombocytes (platelets). In some implementations, the capture particlesbegin binding to the undesirable particles as they are mixed as theyflow down the length of the upstream portion 308. In otherimplementations, the capture particles are mixed with the whole bloodprior to flowing through the first inlet 301, and thus the binding ofthe capture particles and undesirable particles can begin before theblood is flowed through the first inlet 301.

As the fluid in the separation channels flows downstream it passes overa first bulk acoustic transducer 302 and eventually a second bulkacoustic transducer 305. The bulk acoustic transducers 302 and 305impart standing acoustic waves on the separation channels. The standingacoustic waves are transverse to the flow of fluid through theseparation channels. In some implementations, the acoustic bulktransducers 302 and 305 emit standing acoustic waves of differentwavelengths. In other implementations the acoustic bulk transducers 302and 305 emit standing acoustic waves of the same wavelength. Exampleacoustic waves can have, but are not limited to, wavelengths betweenabout 1 and about 4 MHz.

In the network 300, the first acoustic bulk 302 transducer aligns theformed elements, capture particles, and undesirable particles in theinterior of the upstream portions 308 of the separation channels. Afterthe particles in the fluid are aligned in the middle of the flowchannels, the channels angle to align the particles with walls of thedownstream portions 309. With all particles aligned in the middle of theflow channel, fluid substantially away from the middle is free ofcapture particles and undesirable particles. Thus, at the angle in theflow channels, a portion of the fluid, substantially void of formedelements, capture particles and undesirable particles, exits through theupstream outlets 303. The particles continue to flow downstream, nowsubstantially aligned with a wall of the respective downstream portions309. Fluid entering the downstream portions 309 of the separationchannels from the second inlets 304 ensures continued flow of the formedelements and capture particles through the downstream portions of the309 of the separation channels.

In the second stage particles are separated based on the speed at which(and thus distance) they travel from a given aggregation axis towards asecond pressure node induced in the channel by a second standingacoustic wave. As indicated above, due to the angling of the separationchannels, the formed elements and capture particles enter the downstreamportions of the separation channels aggregated along one wall.

As the particles flow along the wall of the separation flow channels,they pass over a second bulk transducer 305, which emits a secondstanding acoustic wave transverse to the flow of the particles. Thesecond standing acoustic wave drives the particles away from the wall.Based on the magnitude of their contrast factor, a first subset ofparticles (e.g., the formed elements of blood) moves away from the wallat a faster rate then a second subset of particles (e.g., the captureparticles).

This differential rate of movement is achieved by using captureparticles that have an acoustophoretic mobility that is substantiallydifferent from that of the formed elements of blood. The differentacoustophoretic mobility is in turn based on the magnitude of thecontrast factor of capture particles being substantially different fromthe magnitude of the contrast factor of the formed elements of blood.The speed at which the two groups move away from the wall can becalculated, and the rate of flow of blood through the system is known.Thus, the distance the formed elements will be moved away from the wallof a separation channel at a given location after being exposed to thesecond acoustic standing wave can also be calculated. This distance istermed d(f, x), where d is the distance traveled away from the wallgiven a specific fluid flow rate (f) and a specific distance (x) afterthe application of the standing acoustic wave. Based on thiscalculation, the separation channel can be divided into two outlets. Thesecond downstream outlet 306 is positioned along the wall the formedelements and capture particles were previously flowing. The seconddownstream outlet 306 is constructed to have a width just smaller thand(f, x). Thus, the formed elements, having traveled a distance of d(f,x) away from the wall due to the second standing acoustic wave wouldhave been driven beyond the second outlet by the time they reach thedistance (x), and thus are driven into the first downstream outlet 307.In contrast, the capture particles bound to the undesirable particles,having traveled a distance substantially less than d(f, x) due to theirlesser acoustophoretic mobility, remain substantially near the wall ofthe downstream portion 309 and exit the second outlet 306. In someimplementations, the distance (x) traveled between exposure to thestanding acoustic wave and entering the first downstream outlet 307 isbetween about 1 and about 10 cm.

FIG. 4 is an illustrative cross-section of a separation channel 400similar to the separation channel depicted in FIG. 2 . The separationchannel 400 includes a top layer 401 sitting atop a bottom layer 402. Achannel is created in the bottom layer 402. When the top layer 401 isplaced on the bottom layer 402 a lumen 403 is created. The separationchannel 400 sits atop a bulk piezoelectric transducer 404. Theseparation channel 400 is secured to the bulk transducer 404, by acoupling adhesive 405 and/or mechanical clamp. In some implementations,the coupling adhesive is cyanoacrylate glue.

The bottom layer 402 and top layer 401 of the separation channel 400 aremanufactured from a substrate sheet. The substrate sheet can be made of,without limitation, polystyrene, glass and polyimide, polyacrylic,polysulfone, and silicon. In some implementations, the bottom layer 402is manufactured by milling, embossing, and/or etching. After creatingthe two layers, they can be joined together by thermacompression,mechanical clamping, adhesive bonding, and/or plasma bonding. Asdescribed above, the separation channel sits atop an acoustic bulktransducer 404. The transducer 404 imparts a standing acoustic wave of aspecific wavelength (λ) across the channel. The dimensions of the bottomlayer 402, top layer 401, and lumen 403 are dependent on the selectedwavelength. In some implementations, the dimensions of the bottom layer402, top layer 401, and lumen 403 are dependent on the selectedwavelength and the material used to manufacture the substrate sheet. Forexample, for substrates formed from glass and some plastics, the widthof the lumen 403 is equal to about half the wavelength (λ_(fluid)/2) ofthe acoustic wave in the fluid. The thickness of the side wall is equalto about a multiple of one quarter of the wavelength (n×λ_(wall)/4) ofthe acoustic wave in the solid channel wall. The height of the lumen ispreferably less than one quarter of the wavelength (<λ_(fluid)/4) of theacoustic wave in the fluid, and the thickness of the top layer 401 canbe arbitrarily selected; however, in some implementations is chosen tobe greater than one quarter of the wavelength (>λ_(wall)/4) of theacoustic wave in the solid channel wall.

In some other implementations, the bottom layer 402, top layer 401, andlumen 403 can have different relative dimensions when differentmaterials are used for the manufacture of the substrate sheet. Forexample, for separation channels 400 formed from a thermoplastic, suchas polystyrene, polyimide, polyacrylic, or polysulfone, the width of thelumen 403 in the bottom layer 402 is less than the one-half thewavelength of the acoustic wave in the fluid. In some implementations tofocus the particles in about the center of the lumen, the width of thelumen 403 in a thermoplastic separation channel 400 is between about 25%and about 45%, about 30% and about 40%, or about 30% and about 35% ofthe wavelength of the acoustic wave in the fluid. The shorter widthresults from the smaller impedance mismatch between the thermoplasticwalls of the separation channel and the fluid passed through thechannel. This lower mismatch provides imperfect acoustic reflection,thereby motivating the narrower channel. Particularly in comparison toglass or silicon-based separation channels, thermoplastic separationchannels are substantially less expensive to manufacture. In someimplementations, when using a thermoplastic, the width of the wall isselected to be between about 35% and about 70%, about 35% and about 50%,about 35% and about 45%, or between about 40% and 45% of the wavelengthof the acoustic wave in the fluid.

In one implementation, a separation channel formed from polystyrene canoperate with an acoustic wave having a frequency of about 1.0 MHz.Assuming the channel is configured for carrying water, the lumen of theseparation channel may be about 0.4 mm wide, or about 40% narrower thanhalf the wavelength of the wave. Moreover, the sidewalls of the bottomlayer 402 of a thermoplastic-based separation channel may besignificantly wider than sidewalls formed from materials that serve asbetter acoustic reflectors. Table 1 below includes additionalexperimental data showing sample thermoplastic separation channeldimensions and excitation frequencies appropriate for carrying eitherHuman Whole Blood (HWB) mixed with Phosphate buffered saline (PBS) or4.4 micron polystyrene (PS) beads suspended in isopropyl alcohol (IPA)at a flow rate of about 100 μL/min. In each of the experiments used tocreate the below table, the top wall was 800 μm thick and the bottomwall was 1000 μm thick. The data set forth in Table 1 is illustrative innature only, and is not intended to narrow the scope of the disclosureincluded herein.

TABLE 1 Channel Width 0.43 0.43 0.53 0.53 0.33 0.97 [mm]: Wall Width[mm]: 1.05 1.05 1 1 1.1 0.78 Device Width [mm]: 2.53 2.53 2.53 2.53 2.532.53 Channel Height 0.2 0.2 0.2 0.2 0.2 0.2 [mm]: Fluid: 20% 4.4 μm 4.4μm 20% 10% HWB 10% HWB HWB PS beads PS beads HWB in PBS in PBS in PBS inIPA in IPA in PBS Frequency Range 0.985-1.9 0.985-1.8 0.66-2.080.66-2.08 0.65-1.8 0.68-1.85 Tested [MHz]: Primary, Single- 1.015 1.0330.92 0.63 1.55 N/A Band Focusing Frequency [MHz] Primary, Dual-Band1.7227 1.76 1.64 1.67 N/A 1.03 Focusing Frequency [MHz] Channel width as30 39 42 23 34 percentage of wavelength in fluid [%] Wall width as 45 4538 26 71 percentage of wavelength in wall material [%]

FIGS. 5A and 5B are cross sectional views of particles suspended in afluid as they flow through a separation channel similar to theseparation channel 200. For FIGS. 5A and 5B, the flow of the fluid istransverse to the plane of the drawings. In some implementations, thefluid is whole blood, and the particles are the formed elements andcapture particles. For illustrative purposes, FIGS. 5A and 5B containstwo particles, red blood cells (white dots) and capture particles (blackdots). FIG. 5A illustrates blood flowing through a channel without astanding acoustic wave being imparted on the channel and its contents.In FIG. 5A, the particles remain homogenously mixed throughout thechannel. In FIG. 5B, a standing wave is imparted on the channel. Thestanding acoustic wave 501 creates two node types. A pressure nodeoccurs at 502. The node extends across the full height of the lumen. Thechannel dimensions set forth above in relation to FIG. 4 yield apressure node at approximately the center of the channel.

Particles are aligned based on the sign of their contrast factor.Particles with a positive contrast factor (e.g. the formed elements ofblood) are driven towards a pressure node 502. In contrast, particleswith a negative contrast factor (e.g. capture particles used in thesingle-stage device described above) are driven toward the pressureantinodes 503.

FIG. 6A is a top view of a separation channel 600, as depicted in FIG. 2, in which fluid is flown through the separation channel 600 without theapplication of the standing acoustic wave. The separation channel 600includes three outlets 602, 603 and 604. As with FIG. 5A, particlessuspended in the fluid are homogeneously distributed throughout thefluid, and thus are not readily discernible in the image. The particlesflow substantially evenly out of all three outlets 602, 603 and 604.

In contrast, FIG. 6B is a top view of the separation channel 600, asdepicted in FIG. 2 , after the application of a standing acoustic wave,according to one illustrative embodiment.

In FIG. 6B, as a result of the standing acoustic wave, the particles 601suspended in the fluid are aligned with the middle of the separationchannel 600. Once aligned with the middle of the separation channel 600,the particles 601 exit the separation channel 600 through the middleoutlet 602. The remaining fluid, substantially devoid of particles,exits the separation channel through the side outlets 603 and 604.

FIG. 7 is an illustrative example of a lipid bilayer capture particle700. The capture particle 700 includes a lipid bilayer 701 encapsulatinga fluid 702. Anchored in the lipid bilayer are affinity particles 703.The affinity particles bind and capture undesirable particles 704.

More specifically, the lipid bilayer 701 forms a liposomal captureparticle. In some implementations, the lipids may be, but are notlimited to dilauroyl-glycero-phosphoglycerol anddilauroyl-glycero-phosphocholine. The capture particle is tuned foracoustically induced mobility. Entities that differ in size, density,and/or compressibility have the greatest differential mobility inacoustic fields and thus are the most readily separable. Therefore, insome implementations, the size, density, and/or compressibility of thecapture particles is modified to distinguish the capture particle fromthe formed elements of blood. The acoustic mobility of a particle isproportional to its volume. For example, in some implementations, thecapture particles are about 1 μm in diameter. In other implementationsthey are between about 2 and about 5 μm in diameter. In some otherimplementations, the capture particles are between about 10 and about 20μm in diameter. In some implementations, use of such larger captureparticles results in a more distinct separation between the captureparticles and red blood cells.

In implementations that adjust the compressibility of the captureparticle, the rigidity of the capture particle can be adjusted bycontrolling the lipid components in the bilayer. The length andsaturation of the lipid hydrocarbon tail, cross-linking of thehydrophobic domains, and/or the inclusion of cholesterol can all affectthe fluidity and compressibility of a liposome.

In other implementations, the density of the liposome is engineered byencapsulating an acoustically active fluid 702. In these implementationsthe acoustical active molecule can be a FDA-approved contrast agent,glycerine, castor oil, coconut oil, paraffin, air, and/or silicone oil.In other implementations, all the above described characteristics aremanipulated to create a capture particle with the greatest possibledifference in contrast factor compared to a formed element.

As described above, the acoustically induced mobility of a particle isbased on the contrast factor of the particle. For a liposomal basedcapture particle, the contrast factor is dominated by the properties ofthe encapsulated fluid. The contrast factor is based on the bulk modulus(K) and density (ρ) of the encapsulated fluid. When suspended in blood,the contrast factor (φ) for a capture particle, encapsulating a specificfluid, is calculated with the below equation:

$\varphi = {\frac{{5\rho} - {2 \cdot 1.02}}{{2\rho} + 1.02} + \frac{2.2}{K}}$

Table 2 provides the ρ, K, and then calculated φ-factor based on theabove equation.

TABLE 2 Calculated Contrast Factors Materials ρ (g/ml) K(Gpa) φEncapsulated glycerine 1.25 4.7 +0.73 Fluids castor oil 1.03 2.06 −0.06coconut oil 0.92 1.75 −0.36 paraffin 0.80 1.66 −0.58 silicone oil 1.041.09 −1.00 air 0.002 1.4 −3.55 Formed white blood cell 1.02 2.5 +0.12Elements red blood cell 1.10 3.0 +0.34

In some implementations, such as the implementation of FIG. 3 , thecapture particles have a contrast factor that is lower in magnitude, butstill of the same sign as the formed elements. In these implementations,the low contrast factor of the capture particles can be achieved bymaking the capture particles sufficiently small to reduce their contrastfactor to below that of the formed elements.

As illustrated in FIG. 7 , affinity molecules 703 are embedded in thelipid bilayer 701. In some implementations, these affinity molecules areglycoconjugates. The glycoconjugates enable the capture and retention ofall major classes of pathogens, including bacteria and viruses. In someimplementations, the affinity molecules 703 also bind to toxins andpro-inflammatory cytokines. In some implementations, affinity molecules703 are designed to universally capture gram-negative and gram-positivebacteria, virus, toxins, by exploiting that: 1) pathogens expressunusual surface N- and O-linked glycan structures that can be targetedby glycan-binding proteins or lectins and 2) many pathogens and toxinsbind to charged polysaccharides, especially those of the heparan sulfatefamily, that are present on the cell surface of mammalian cells. Someimplementations employ glycoconjugate capture agents that have twocomponents: a modified, non-anticoagulant heparin fragment thatnevertheless maintains high affinity, multivalent binding properties,and a glycan-binding protein that binds to surface N- and O-linkedglycans present on the surface of pathogens. In other implementations,the glycan structure is a lectin. For example the lectin can be, but isnot limited to: type 2 membrane receptors such as DC-SIGN, DC-SIGNR, andLangerine; collectins such as pulmonary surfactant proteins (SP-D,SP-Al), mannose binding lectin, and collectin-Kl; and macrophage mannosereceptors. In other implementations, the affinity molecule is anantibody.

The affinity particles are anchored to the liposomal surface so theirconcentration, valency, and distribution can be controlled. This isparticularly relevant since pathogen-receptor interactions are oftenmultivalent and the receptor configuration impacts overall avidity. Insome implementations, the affinity molecule is attached to an anchorthat is incorporated into the lipid bilayer, so the embedded functionalgroups remain in close proximity but are free to rotate and rearrange.Lipid anchors are favored because the molar ratio of derivatized lipidsincorporated can be controlled. Lectins are incorporated by solubalizinga surfactant with pre-formed liposome suspensions, through directaddition of fatty acids to lysine residues, or by modification withhydrophobic anchor lipids such as Nglutaryl-phosphotidylethanolamine(NGPE).

FIG. 8 illustrates an overview of the process of making and using acapture particle. The affinity molecules of FIG. 8A are embedded in theliposome of FIG. 8B to produce a affinity coated liposome as illustratedin FIG. 8C. Next, the capture particle is combined with a blood or otherfluid containing undesirable particles. The undesirable particles thenbind to the capture particles. FIG. 8E illustrates, bound undesirableparticles can then be removed from the fluid by acoustically moving thecapture particles whereas unbound undesirable particle are not removedfrom the fluid.

FIG. 9 is a flow chart of a method 900 to cleanse blood from undesirableparticles in a blood cleaning separation channel similar to thetwo-stage device described in FIG. 3 . First whole blood is flowedthrough a microfluidic separation channel (step 901). Capture particlesare introduced into the whole blood (step 902). Then, formed elements ofthe blood and the capture particles are directed away from the walls ofthe separation channel (step 903). Next, the capture particles andformed elements are directed alongside a wall of the separation channel(step 904). Then, formed elements are driven away from the walls of theseparation channel (step 905). Finally, the formed elements arecollected in a first downstream outlet and the capture particles arecollected in a second downstream outlet (steps 906 and 907,respectively).

Referring to FIGS. 3 and 9 , the method of cleansing blood includesflowing whole blood through a microfluidic separation channel (step901). The whole blood contains plasma; a plurality of formed elementssuch as red blood cells, white blood cells, and platelets; andundesirable particles. In some implementations, the whole blood isflowed through a plurality of microfluidic separation channels, such asthe network of channels 300, connected to one another by a manifoldsystem, while in other implementations a single separation channel isused. In some implementations, the whole blood is extracted from apatient. In other implementations the whole blood is collected from apatient or donor and stored prior to cleansing.

Capture particles are introduced into the whole blood (step 902). Insome implementations, the capture particles are introduced into thewhole blood at the first inlet 301 of the separation channel depicted inFIG. 3 . In other implementations, the capture particles are introducedinto the device's manifold system or in a mixing chamber upstream fromthe manifold. After introduction into the whole blood, the captureparticles begin to bind to the undesirable particles in the whole blood.For example, a capture particle configured to remove a specific bacteriafrom the whole blood will selectively bind to the bacteria.

Next, the method 900 continues with the capture particles and the formedelements of the whole blood, which are, originally, substantially-evenlydispersed throughout the whole blood, being directed away from the wallsof the separation channel (step 903) and aggregated into alignment atabout the center of the separation channel. In some implementations,such as the implementation in the network 300 as described above, thisis done with a first bulk transducer 302 imparting a standing acousticwave across the channel transverse to the direction of flow within thechannel in an upstream portion 308, resulting in a pressure node atabout the center of the separation channel. In such implementations thecontrast factor of the capture particles has the same sign as that ofthe formed elements, thus the formed elements and capture particles movein tandem towards the pressure node. This initial aggregation ofparticles along a common axis enables later separation of the captureparticles from the formed elements of blood due based on theirdifferential acoustophoretic mobilities.

As discussed above in reference to network 300, after an initialaggregation (step 903), the method 900 continues with the captureparticles and formed elements of blood being directed alongside adownstream wall of the separation channel (step 904). In someimplementations, such as that of network 300, this is accomplished by ashift in the separation channel such that the downstream portion of theseparation channel is significantly aligned with the middle of theupstream portion of the separation channel.

As depicted in FIG. 3 above, in the downstream portion 309, the method900 continues with the formed elements being driven way from the wallsof the separation channel (step 905). As mentioned above, in someimplementations, the contrast factor of the formed elements and thecapture particles have the same sign but are of different magnitudes.Thus, the formed elements will migrate away from the wall at a fasterrate than the capture particles and undesirable particles. In otherimplementations, the capture particles are designed to have a contrastfactor magnitude larger than the formed elements of blood, thus thecapture particles move away from the wall at a faster rate than theformed elements.

In some implementations, the standing waves applied to the upstreamportion 308 and/or to the downstream portion 309 are periodically haltedfor a limited amount of time. Doing so allows capture particles orformed elements that may have become trapped against a sidewall of theseparation channel 300 to be released, thereby preventing clogging orcongestion in the channel. For example, for devices utilizing anexcitation frequency of about 1.0 MHz, the standing waves may be haltedabout once every second for about one tenth of second. In otherimplementations, the standing waves may be halted more or lessfrequently with shorter or longer durations depending, for example, onthe length and width of the channel and the flow rate of fluid throughthe channel. In general, the standing wave has a duty cycle of betweenabout 75% and about 98%.

The method 900 concludes when the formed elements are collected in afirst downstream outlet (step 906) and the capture particles beingcollected in a second downstream outlet (step 907). As described abovein relation to network 300, the second downstream outlet 306 isconfigured to collect fluid containing capture particles substantiallydevoid of formed elements. In some implementations, this is achieved byconfiguring the width of the second downstream outlet 306 to be slightlyless than d(f, x), the distance the formed elements travel in responseto the standing acoustic wave given a flow rate off and a distance xfrom the point of application of the standing acoustic wave. Thus theformed elements, having been driven d(f, x) away from the of theseparating channel will be collected in the first downstream outlet 307.

FIG. 10 is a flow chart of a method for cleansing blood with asingle-stage microfluidic separation channel (1000). First, whole bloodis collected (step 1001). Then whole blood is flowed into an inlet of asingle-stage microfluidic separation channel, as depicted in FIG. 2(step 1002). Next, a plurality of capture particles is introduced intothe whole blood (step 1003). Then a standing acoustic wave is applied tothe separation channel (step 1004). The formed elements are thencollected in a first outlet (step 1005). Next, the capture particles arecollected in a second outlet (step 1006). Finally, the cleansed blood isreturned to a storage container or returned directly to the patient(step 1007).

Referring to FIGS. 1, 2 and 10 , the method 1000 of cleansing blood witha single-stage microfluidic separation channel 200 begins by collectingwhole blood. In some implementations, the whole blood is collected froma patient 101, and then directly introduced into the blood cleansingsystem 100. In other implementations, the whole blood is collected froma patient 101 and then stored for later cleansing.

Next, the method 1000 of cleansing blood with a single-stagemicrofluidic separation channel 200 continues by flowing whole bloodinto the inlet of a microfluidic separation channel (step 1002). Thewhole blood contains a plurality of formed elements, plasma, and aplurality of undesirable particles. In some implementations, theundesirable particles can be toxins, bacteria, and/or viruses. In someimplementations, a single microfluidic separation channel is used, whilein others a plurality of single-stage separation channels is used inconjunction to accommodate greater blood flow throughput.

The method 1000 continues with the introduction of a plurality ofcapture particles into the whole blood (step 1003). In someimplementations, the constituent components of capture particles areinjected into a separation channel with a micronozzle and spontaneouslyform capture particles as injected into the separation channel. In otherimplementations, the capture particles are prefabricated and thenintroduced into the whole blood. In some implementations, the captureparticles are introduced into the whole blood after the whole bloodenters the separation channel through the first inlet 202. In yet otherimplementations, the capture particles are introduced into the wholeblood before the blood enters through the first inlet 202 of theseparation channel 200. In some implementations, the capture particlesare microbeads and/or lipid based liposomes.

Next, the method 1000 continues with the applying of a standing acousticwave to the separation channel (step 1004). The standing acoustic waveis applied transverse to a direction of flow of the whole blood throughthe separation channel 200. In some implementations, the formed elementsand capture particles have contrast factors with different signs. Thus,the application of the standing acoustic wave causes the formed elementsto aggregate about the central axis of the separation channel and thecapture particles to aggregate along at least one wall of the separationchannel, as depicted in FIG. 2 . In other implementations the standingacoustic wave causes the formed elements to aggregate along at least onewall of the separation channel and the capture particles to aggregateabout the central axis of the separation channel.

In some implementations, the standing wave is periodically halted for alimited amount of time. Doing so allows capture particles or formedelements that may have become trapped against a sidewall of theseparation channel 200 to be released, thereby preventing clogging orcongestion in the channel. For example, for devices utilizing anexcitation frequency of about 1.0 MHz, the standing wave may be haltedabout once every second for about one tenth of second. In otherimplementations, the standing wave may be halted more or less frequentlyor for shorter or longer durations depending, for example, on the lengthand width of the channel and the flow rate of fluid through the channel.In general, the standing wave has a duty cycle of between about 75% andabout 98%.

Then, the method 1000 continues with the collecting of the formedelements of the whole blood in a first outlet (step 1005). In someimplementations, as depicted in FIG. 2 , a first outlet 204 is alignedwith the central axis of the separation channel allowing the outlet tocollect the formed elements as they aggregate and flow down the centralaxis of the separation channel. Similarly, the method continues with thecollecting of the capture particles in a second outlet (step 1006). Insome implementations, the end of the separation channel has at least asecond outlet channel 206 and 207 aligned with at least one wall of theseparation channel. As the capture particles are driven towards theantipressure notes along the walls of the separation channel, they arecollected by the outlets channels 206 and 207 aligned with the walls ofthe separation channels. In some implementations, the standing acousticwave is adjusted such that the formed particle align along the walls ofthe separation channel and the capture particles align with the centralaxis of the separation channel. In such an implementation, the formedelements are funneled into outlets along the wall of the separationchannel and the capture particles are funneled into an outlet alignedwith the central axis of the separation channel. In someimplementations, the outlet channels 206 and 207 terminate in individualoutlets or merge to terminate into a single outlet 205.

The method 1000 concludes with the reintroduction of the cleansed bloodinto a patient 101 or storage (step 1007). In some implementations, suchas system 100, the whole blood is collected directly from a patient andthen reintroduced to the patient 101. In some implementations, thecleansed blood is reheated to body temperature before being reintroducedinto the patient 101. In other implementations, the cleansed blood iscollected in a storage container for later reintroduction into a patient101.

What is claimed is:
 1. A blood cleansing device comprising: amicrofluidic separation channel defined in a thermoplastic and having anupstream end and downstream end, the microfluidic separation channelcomprising: a first inlet configured to introduce flowing whole bloodinto a proximal end of the microfluidic separation channel, the wholeblood including plasma, a plurality of formed elements and a pluralityof undesirable particles; a first outlet at the downstream end of themicrofluidic separation channel positioned substantially along alongitudinal axis of the microfluidic separation channel; a secondoutlet at the downstream end positioned adjacent a first wall of themicrofluidic separation channel; and an acoustic transducer positionedadjacent to the microfluidic separation channel and configured tooperate at a predetermined frequency to generate a standing acousticwave across a particle migration region of the microfluidic separationchannel, wherein a width of the microfluidic separation channel isbetween about 25% and 35% of an acoustic wavelength in the whole bloodat the predetermined frequency.
 2. The blood cleansing device of claim1, wherein the width of the microfluidic separation channel is betweenabout 30% and about 35% of the acoustic wavelength in the whole blood atthe predetermined frequency.
 3. The blood cleansing device of claim 1,wherein a thickness of a wall material of the microfluidic separationchannel is between about 35% and about 45% of the acoustic wavelength inthe wall material at the predetermined frequency.
 4. The blood cleansingdevice of claim 1, wherein the standing acoustic wave is generated in adirection substantially transverse to the longitudinal axis of themicrofluidic separation channel.
 5. The blood cleansing device of claim1, further comprising a capture particle injector configured tointroduce a plurality of lipid-based capture particles into the wholeblood before the whole blood reaches the particle migration region ofthe microfluidic separation channel.
 6. The blood cleansing device ofclaim 5, comprising a reservoir in fluidic communication with thecapture particle injector.
 7. The blood cleansing device of claim 6,wherein the reservoir contains the plurality of lipid-based captureparticles.
 8. The blood cleansing device of claim 6, wherein thereservoir contains a mixture of materials, which when directed by thecapture particle injector into the whole blood, form the plurality oflipid-based capture particles.
 9. The blood cleansing device of claim 8,wherein the mixture of materials comprises an affinity molecule, alipid, and a fluid with a density less than about 1 g/cm3.
 10. The bloodcleansing device of claim 5, wherein the plurality of lipid-basedcapture particles have significantly different acoustophoretic mobilitythan that of formed elements of blood.
 11. The blood cleansing device ofclaim 5, wherein the capture particle injector comprises a microfluidicnozzle.
 12. The blood cleansing device of claim 5, wherein the captureparticle injector comprises a porous membrane.
 13. The blood cleansingdevice of claim 1, wherein the second and third outlets merge at afourth outlet.